Oxo-M and 4-PPBP induction of tenogenic differentiation of perivascular tendon stem cells

ABSTRACT

Provided herein are compositions including oxotremorine (e.g., oxotremorine methiodide or Oxo-M) and 4-PPBP (e.g., 4-PPBP maleate). Also provided are methods of treating a connective tissue defect in a subject with oxotremorine and 4-PPBP. In addition, provided are scaffolds and methods of making same that include multiple fibers that include Oxo-M, 4-PPBP, and optionally icariin or kartogenin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part from PCT/US17/22751 filedMar. 16, 2017 which claims the benefit of U.S. Provisional ApplicationSer. No. 62/309,050, filed Mar. 16, 2016 and claims priority under 35USC 119 and 120 to the foregoing applications.

The present application also claims the benefit of U.S. ProvisionalApplication No. 62/668,582 filed May 8, 2018, which are incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

Current stem cell-based strategies for tissue regeneration involve exvivo manipulation of these cells to confer features of the desiredprogenitor population. Despite being a valid approach, celltransplantation has encountered crucial barriers in therapeutictranslation, including immune rejection; pathogen transmission;potential tumorigenesis; issues associated with packaging, storage, andshipping; and difficulties in clinical adoption and regulatory approval.

Perivascular tendon stem/progenitor cells (PTSCs) are a small populationof multipotent cells that play a critical role in tendon healing andregeneration. While connective tissue growth factor (CTGF) has beenshown to induce PTSCs to differentiate into tendon cells and helps torecruit PTSCs to the site of injury following tendon rupture, theclinical use of CTGF as a therapy for tendon injury may be limited bythe many challenges typically associated with biologics.

CTGF is known to recruit CD146+ perivascular tendon stem/progenitorcells (PTSCs) to the site of injury following tendon rupture; inducedifferentiation of PTSCs into tendon cells; and regenerate fullytransected tendons (Lee, et al., J. Clin. Invest. 125:2690-2701, 2015).CTGF-induced proliferation and tenogenic differentiation of PTSCs areregulated by the focal adhesion kinase (FAK) and extracellularsignal-regulated kinase (ERK) 1/2 pathway (Lee, et al., 2015, supra),consequently leading to tendon regeneration in rats.

CTGF can selectively enrich PTSCs in the early phase of tendon healingand induce tenogenic differentiation in the later phase (Lee, et al.,2015, supra). Localized delivery of CTGF can promote healing andregeneration of transected tendons in vivo by inducing a transientincrease in PTSCs (Lee, et al., 2015, supra).

The PTSCs are a small population of multipotent cells that play acritical role in tendon healing and regeneration, and the stimulation oftenogenic differentiation of PTSCs is a key to regenerate torn tendons(see, e.g., Lee, et al., 2015, supra). PTSCs can be found in theperivascular niche and express both tendon- and stem cell-likecharacteristics (Tempfer, et al., Histochem. Cell Biol. 131:733-741,2009; Lee, et al., 2015, supra).

Despite these promising results, however, the clinical use of CTGF as atherapy for tendon injury may be limited by the many challengestypically associated with biologics, which include high cost,immunogenicity, and the necessity for complex delivery systems.

Oxotremorine M (Oxo-M) and PPBP maleate (4-PPBP) are receptor agonistswhich activate the same signaling pathway that regulates CTGF-mediatedtenogenic differentiation of PTSCs. Oxo-M has been identified as amuscarinic receptor agonist that can elicit FAK signaling in neuronalcells (Linseman, et al., J. Neurochem. 73:1933-1944, 1999).4-phenyl-1-(4-phenylbutyl) piperidine (4-PPBP) is a small molecule σ₁receptor agonist that can elicit ERK1/2 phosphorylation in primaryneurons (Tan, et al., Neuropharmacology 59:416-424, 2010).

SUMMARY OF THE INVENTION

A combination of two small molecule compounds oxotremorine (Oxo-M) andPPBP (4-PPBP maleate), have been shown to induce differentiation ofPTSCs into tendon-like cells. These compounds activate the samesignaling pathway that regulates CTGF-mediated differentiation of PTSCsand, when combined, can be more effective than even CTGF at increasingexpression of tendon markers. This combinatorial small molecule compoundapproach can be adapted for tendon or ligament therapy or for softtissue research applications.

Studies described herein show the combination of Oxo-M and 4-PPBP caninduce tenogenic differentiation of PTSCs; Oxo-M significantly increasesPTSC expression of tenogenic markers tenascin-C, vimentin, and scleraxisafter 1 week of treatment in vitro; 4-PPBP significantly increases PTSCexpression of tenogenic markers collagen I and III after 1 week oftreatment in vitro; and the combined administration of Oxo-M and 4-PPBPhas synergistic effects on tenogenic differentiation and increasesexpression of tenogenic markers collagen I and II, vimentin,tenomodulin, and scleraxis to comparable or higher levels than CTGFstimulation.

One aspect provides a composition. In some embodiments, the compositionincludes oxotremorine (e.g., oxotremorine methiodide, Oxo-M) and PPBP(e.g., 4-PPBP maleate). In certain embodiments, the concentration ofOxo-M is between about 10 μM and about 100 mM, or between about 100 μMand about 10 mM. In particular embodiments the concentration of Oxo-M isabout 1 mM. In other embodiments the concentration of 4-PPBP is betweenabout 100 nM and about 1 mM, or between about 1 μM and about 100 μM. Inyet other embodiments the concentration of 4 PPBP is about 10 μM. Infurther embodiments the concentration of Oxo-M is about 1 mM and theconcentration of 4 PPBP is about 10 μM. In some embodiments, thecomposition is a pharmaceutical composition including oxotremorine or asalt thereof (e.g., Oxo-M), 4-PPBP or a salt thereof (e.g., 4-PPBPmaleate), and a pharmaceutically acceptable excipient. In someembodiments, the composition or pharmaceutical composition furtherincludes a matrix material, a surgical adhesive, or a fibrin glue. Inadditional embodiments the composition or pharmaceutical compositionfurther includes an antibiotic, an anti-inflammatory, a cytokine, agrowth factor, or a stem cell. In still other embodiments the Oxo M andsaid 4-PPBP are encapsulated in a microsphere.

Another aspect provides a method of treating a connective tissue defectin a subject. In some embodiments, the method includes administeringoxotremorine or a salt thereof (e.g., Oxo-M) and 4-PPBP or a saltthereof (e.g., 4-PPBP maleate) to a subject in need thereof. In someembodiments, the method includes providing a pharmaceutical compositioncomprising an effective amount, or a therapeutically effective amount,of oxotremorine or a salt thereof (e.g., Oxo-M), an effective amount, ora therapeutically effective amount, of 4-PPBP or a salt thereof (e.g.,4-PPBP maleate), and a pharmaceutically acceptable excipient: andadministering the composition to the connective tissue defect of thesubject; wherein the connective tissue defect is a tendon injury or aligament injury. In certain embodiments the connective tissue defect isa tendon injury, for example a patellar tendon injury, an Achillestendon injury, a rotator cuff injury, or lateral epicondylitis. In someembodiments the composition further includes a matrix material, asurgical adhesive, or a fibrin glue. In additional embodiments thecomposition or pharmaceutical composition further includes anantibiotic, an anti-inflammatory, a cytokine, a growth factor, or a stemcell. In still other embodiments the Oxo M and said 4-PPBP areencapsulated in a microsphere. In particular embodiments the subject isa human subject.

Other objects and features will be in part apparent and in part pointedout hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1A-1F is a series of bar graphs showing tendon-related geneexpressions in CD146+ perivascular tendon stem/progenitor cells (PTSCs)when treated by selected small molecules. At 1 week of treatment, 4-PPBPsignificantly increased COL-I (FIG. 1A) and COL-III (FIG. 1B), whereasOxo-M significantly increased Tn-C (FIG. 1C), VIM (FIG. 1D), and Scx(FIG. 1F) (*: p<0.05 compared to control). Oxo-M: Oxotremorine M (1 mM),PAO: Phenylarsine oxide (2 μM), 4-PPBP: PPBP maleate (10 μM), BOM:[Tyr4]-Bombesin (10 nM).

FIG. 2A-2F is series of bar graphs showing tendon-related geneexpressions in CD146+ perivascular tendon stem/progenitor cells (PTSCs)when treated by Oxo-M+4-PPBP. At 1 week of treatment, Oxo-M+4-PPBPsignificantly increased expressions of COL-I (FIG. 2A), III (FIG. 2B),Tn-C (FIG. 2C), VIM (FIG. 2D), TnmD (FIG. 2E), and Scx (FIG. 2F), at asimilar or higher level of CTGF (*: p<0.05 compared to control; #:p<0.05 compared to 100 ng/mL CTGF).

FIG. 3A and FIG. 3B are a Western Blot showing phosphorylation of FAKand ERK1/2 induced by Oxo-M and 4-PPBP (FIG. 3A) and siRNA knockdown ofFAK and ERK1/2 significantly reduced Scx expression induced by Oxo-M and4-PPBP (FIG. 3B). n=5 per group; *: p<0.05 compared to all the othergroups.

FIG. 4A-4H is a series of H&E stained slides showing healing of fullytransected rat patellar tendons by small molecules. At 1 week post-op,delivery of Oxo-M (1 mM)+4-PPBP (10 μM) resulted in reduced gaps andmore aligned collagen structure (FIG. 4D), as compared to disorganizedscar-like tissue with control (FIG. 4A), Oxo-M alone (FIG. 4B), and4-PPBP alone (FIG. 4C). At 2 weeks post-op, control (FIG. 4E), Oxo-Malone (FIG. 4F), and 4-PPBP alone (FIG. 4G) groups ended up withscar-like healing, whereas Oxo-M+4-PPBP (FIG. 4H) showed significantlyimproved healing with densely aligned fibers.

FIG. 5A-5H shows Masson's Trichrome staining of healing zone of tendons.Delivery of Oxo-M (1 mM)+4-PPBP (10 μM) showed more and denser collagenin the healing zone by 2 weeks post-op (FIG. 5H) as compared todisrupted collagen structure with control (FIG. 5E), Oxo-M alone (FIG.5F), and 4-PPBP alone (FIG. 5G) delivered groups. No major differenceswere seen between the groups at 1 week post-op (FIG. 5A-D).

FIG. 6A-6H shows polarized image of Picrosirius Red (PR) stained slides.Delivery of Oxo-M (1 mM)+4-PPBP (10 μM) showed re-organized collagenfibers by 1 week post-op (FIG. 6D) and 2 weeks post-op (FIG. 6H) ascompared to disrupted collagen structure at 1 week and 2 weeks post-opwith control (FIG. 6A and FIG. 6E, respectively), Oxo-M alone (FIG. 6Band FIG. 6F, respectively), and 4-PPBP alone (FIG. 6C and FIG. 6G,respectively) delivered groups.

FIG. 7A-7D shows immunofluorescence for CD146 and COL-I in tendonhealing by control (FIG. 7A), Oxo-M alone (FIG. 7B), 4-PPBP alone (FIG.7C) and a combination of Oxo-M and 4-PPBP (FIG. 7D) at 1 week post-op.

FIG. 8A-8F shows collagen fiber orientation assessed by Picrosirius Redstaining with an automated digital image processing for localdirectionality and angular deviation (AD). Alignment of collagen fibersin the Oxo-M+4-PPBP delivered tendon (FIG. 8D) was similar to that ofthe native tendon (FIG. 8E), in contrast to disoriented fibers inscar-like tendon with control (FIG. 8A), Oxo-M alone (FIG. 8B), and4-PPBP alone (FIG. 8C). Quantitatively, the AD of fibers withOxo-M+4-PPBP was significantly smaller than all the other groups (FIG.8F). n=6 per samples, *: p<0.001 compared to control, Oxo-M, and 4-PPBP,#: p<0.05 compared to Oxo-M+4-PPBP.

FIG. 9A-9I is a series of H&E stained slides showing healing oftransected rat supraspinatus tendons by Oxo-M plus 4-PPBP at 4×, 1033and 20× magnification at 4 weeks post-op. The combination of Oxo-M and4-PPBP shows repair of transected rat supraspinatus tendons (FIG. 9C,FIG. 9F and FIG. 9I) to more closely resemble native rat supraspinatustendons (FIG. 9A, FIG. 9D and FIG. 9G) compared to control (FIG. 9B,FIG. 9E and FIG. 9H).

FIG. 10A-10I is a series of Picrosirius Red stained slides showinghealing of transected rat supraspinatus tendons by Oxo-M plus 4-PPBP at4×, 1033 and 20× magnification at 4 weeks post-op. The combination ofOxo-M and 4-PPBP shows repair of transected rat supraspinatus tendons(FIG. 100, FIG. 10F and FIG. 10I) to more closely resemble native ratsupraspinatus tendons (FIG. 10A, FIG. 10D and FIG. 10G) compared tocontrol (FIG. 10B, FIG. 10E and FIG. 10H).

FIG. 11A-11I is a series of Masson's Trichrome stained slides showinghealing of transected rat supraspinatus tendons by Oxo-M plus 4-PPBP at4×, 10× and 20× magnification at 4 weeks post-op. The combination ofOxo-M and 4-PPBP shows repair of transected rat supraspinatus tendons(FIG. 11C, FIG. 11F and FIG. 11I) to more closely resemble native ratsupraspinatus tendons (FIG. 11A, FIG. 11D and FIG. 11G) compared tocontrol (FIG. 11B, FIG. 11E and FIG. 11H).

FIG. 12A-12I is a series of Safranin O stained slides showing healing oftransected rat supraspinatus tendons by Oxo-M plus 4-PPBP at 4×, 10× and20× magnification at 4 weeks post-op. The combination of Oxo-M and4-PPBP shows repair of transected rat supraspinatus tendons (FIG. 12C,FIG. 12F and FIG. 12I) to more closely resemble native rat supraspinatustendons (FIG. 12A, FIG. 12D and FIG. 12G) compared to control (FIG. 12B,FIG. 12E and FIG. 12H).

FIG. 13 shows microencapsulation conditions and design parameters asdetermining factors of EE and release rate (RR) for PLGA microspherescomprising Oxo-M and 4-PPBP.

FIG. 14 is an illustration showing a scaffold fabricated with strandshaving Oxo-M+4-PPBP, Oxo-M+4-PPBP+Kartogenin, and Icariin embeddedtherein.

FIG. 15 is a series of images showing the implementation of visualagents (e.g. quantum dots) that allow for the tracking of in vivodegradation of implanted polymeric scaffolds.

FIG. 16 is a series of images that show 3D printed scaffolds thatpossess anatomical shape/dimension of tissues, native-like internalmicrostructures with matched mechanical properties, provide forspatiotemporal delivery of small molecules (e.g. Oxo-M, 4-PPBP,kartogenin and icariin), and labeled with a visual agent (e.g. quantumdots). The scaffold can be visualized with near infrared imaging.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the discovery thata combination of oxotremorine M (Oxo-M) and PPBP (4-PPBP maleate)induces tenogenic differentiation of perivascular tendon stem cells. Thecombination of Oxo-M and 4-PPBP, which has been shown to inducedifferentiation of PTSCs into tendon-like cells, can be used as atherapy for healing or regenerating tendon injury or ligament injury.

As shown herein, healing, repair, or regeneration can be achieved byrecruiting, activating, or differentiating either tissue-resident orcirculating stem cells, instead of stem cell transplantationnecessitating ex vivo manipulation. Regeneration by harnessing theregenerative potential of endogenous stem cells can serve as astraightforward strategy for regenerative medicine that can overcome thecurrent translational hurdles associated with cell transplantation. Thusis provided a breakthrough in treatment of injured or degeneratedtissues, tendon, or ligament.

This discovery provides for use of small molecule chemical compounds asan alternative or supplement to conventional growth factor approachesfor induction of tenogenic differentiation of tendon stem cells andrelated compositions and methods for tendon and ligament healing orregeneration.

As shown herein, a combination of two small molecule chemical compounds,Oxotremorine M (Oxo-M) and PPBP maleate (4-PPBP) induces the tenogenicdifferentiation of CD146+ perivascular tendon stem/progenitor cells(PTSCs) with a similar or higher level of efficiency compared to CTGF.First tested were several selected FAK or ERK1/2 agonists, includingOxo-M, 4-PPBP, Phenylarsine oxide (PAO), and [Tyr4]-Bombesin (BOM), tocultured rat PTSCs. Results described herein showed that after 1 week invitro, only Oxo-M (1 mM) and 4-PPBP (10 μM) resulted in 1.5-2.5 foldincrease in tendon-related gene expressions, including collagen I andIII, tenascin-C (Tn-C), vimentin (VIM), tenomodulin (Tnmd), andscleraxis (Scx). Further experiments showed when Oxo-M (1 mM) and 4-PPBP(10 μM) were applied together, the tendon-related gene expressions weredramatically increased up to about 6-40 fold at a similar level achievedby CTGF. These findings show that Oxo-M and 4-PPBP have synergeticeffects to induce tenogenic differentiation of the specific populationof tendon stem/progenitor cells. It was previously shown that thatstimulation of tenogenic differentiation of PTSCs is a key to regeneratetorn tendons (see, e.g., Lee, et al., 2015, supra). New findings that acombination of Oxo-M and 4-PPBP dramatically increase (e.g.,synergistically increase) tendon-related gene expressions provideseffective therapeutic compositions and methods for treatment of tendoninjuries.

In some embodiments, Oxo-M and 4-PPBP each induce expression oftendon-related markers in PTSCs. In some embodiments, the synergisticcombination of Oxo-M and 4-PPBP can further increase expression of keytendon markers to comparable or higher levels than CTGF stimulation.This combination of Oxo-M and 4-PPBP can be used as a smallmolecule-based therapeutic for stem cell-mediated repair of tendon andligament injuries.

The combination of chemical compounds described herein can provide abetter route of administration or well-controlled outcome.

One embodiment of the present disclosure provides a pharmaceuticalcomposition for treating tendon or ligament injury in a subject (e.g.,an animal or human). One embodiment of the present disclosure provides apharmaceutical composition for incorporation into a graft or patch topromote tendon healing in in a subject. One embodiment of the presentdisclosure provides for generation of tenocytes from stem cells fortendon tissue engineering. One embodiment of the present disclosureprovides a tool to study molecular mechanisms of tenogenesis and tendondevelopment.

Oxo-M

Oxotremorine (CAS No. 70-22-4; ChEMBL7634; C₁₂H₁₈N₂O) has the followingstructure:

Oxotremorine is also known as1-(4-[1-Pyrrolidinyl]-2-butynyl)-2-pyrrolidinone;1-(4-[1-Pyrrolidinyl]2-butynyl)-2-pyrrolidinone; or1-(4-Pyrrolidin-1-ylbut-2-yn-1-yl)pyrrolidin-2-one.

Oxotremorine is available as a salt. Oxotremorine methiodide (Oxo-M) isthe iodine salt of Oxo.

Oxotremorine sesquifumarate salt has the following structure:

Oxotremorine and salts thereof are commercially available (see, e.g.,Tocris Bioscience; Sigma-Aldrich).

The present disclosure refers to Oxo-M but it is understood that suchrecitation can refer to oxotremorine or another salt form ofoxotremorine.

4-PPBP

PPBP (4-PPBP) (CAS Number 136534-70-8; 4-phenyl-1-(4-phenylbutyl)piperidine; C₂₁H₂₇N) has the following structure:

PPBP maleate (4-PPBP maleate) (CAS Number 207572-62-1;4-phenyl-1-(4-phenylbutyl)-piperidine maleate; C₂₁H₂₇N.C₄H₄O₄) has thefollowing structures:

Maleate is the ionized (i.e., deprotonated) form of maleic acid and willhave a negative charge. In the diagram below, X is understood as ahalogen salt (e.g., NaCl, CaCl₂ or a similar salt).

The pH can be adjusted to deprotonate. The pKa of maleic acid is 2,which is low enough to where it would easily give up a proton to a base(e.g., NaOH, Ca(OH)₂)

PPBP maleate is commercially available (see, e.g., Tocris Bioscience;Sigma-Aldrich; Santa Crux Biotech).

The present disclosure refers to 4-PPBP but it is understood that suchrecitation can refer to 4-PPBP or 4-PPBP maleate or another salt form.

Kartogenin

Kartogenin has the following structure:

Kartogenin is available from Stem Cell Technologies (Cat #72572). Thepresent disclosure refers to Kartogenin but it is understood that suchrecitation can refer to kartogenin or a salt form thereof.

Icariin

Icariin has the following structure:

Icariin is available from multiple sources including LiveLong Nutrition(Cat #B004579RJY). The present disclosure refers to icariin but it isunderstood that such recitation can refer to icariin or a salt formthereof.

Progenitor Cells

A progenitor cell, as that term is used herein, is a precursor to afibrochondrocyte or fibrochondrocyte-like cell and can differentiate inthe presence of CTGF or a composition described herein. A progenitorcell can be a multipotent cell. A progenitor cell can be self-renewing.For example, a progenitor cell can be a mesenchymal stem cell (e.g., ahuman mesenchymal stem cell). The progenitor cell can be substantiallyless differentiated than a fibrochondrocyte or fibrochondrocyte-likecell. The progenitor cell can be a perivascular tendon stem/progenitorcell (PTSC). The progenitor cell can be a CD146+ perivascular tendonstem/progenitor cell.

Chemistry

The following definitions and methods are provided to better define thepresent disclosure and to guide those of ordinary skill in the art inthe practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

A chemical bond is understood as an attraction between atoms of abiomolecule and atoms of a matrix material that allows the formation ofa linkage between atoms (e.g., atoms of the same molecule or differentmolecules). A bond can be caused by an electrostatic force of attractionbetween opposite charges, either between electrons and nuclei, or as theresult of a dipole attraction. A bond can be, for example, a covalentbond, a coordinate covalent bond, an ionic bond, polar covalent, adipole-dipole interaction, a London dispersion force, a cation-piinteraction, or hydrogen bonding.

Encapsulation

As described herein, an active agent can be encapsulated in amicrosphere (μS). Such microspheres are useful as slow releasecompositions. For example, small molecules such as Oxo-M, 4-PPBP,kartogenin and/or icariin can be micro-encapsulated to provide forenhanced stability or prolonged delivery. Encapsulation vehiclesinclude, but are not limited to, microparticles, liposomes,microspheres, or the like, or a combination of any of the above toprovide the desired release profile in varying proportions. Othermethods of controlled-release delivery of agents will be known to theskilled artisan. Moreover, these and other systems can be combined ormodified to optimize the integration/release of agents. For example, theagents encapsulated in the microspheres can have a total accumulatedrelease rate of from about 0% to about 50% over the course of at least42 days. It is understood that recitation of the above range includesdiscrete values between the recited range. One skilled in the art willunderstand that the distribution of release rate can have anydistribution including a normal distribution or a non-normaldistribution.

For example, the polymeric delivery system can be a polymericmicrosphere, e.g., a PLGA polymeric microsphere. A variety of polymericdelivery systems, as well as methods for encapsulating a molecule suchas a growth factor, are known to the art (see, e.g., Varde and Pack,Expert Opin. Biol. Ther. 4:35-51, 2004). Polymeric microspheres can beproduced using naturally occurring or synthetic polymers and areparticulate systems in the size range of 0.01 to 500 μm. Polymericmicrospheres can have a mean diameter of about 0.01 μm, about 0.05 μm,about 0.1 μm, about 0.5 μm, about 1 μm, 10 μm, about 20 μm, about 30 μm,about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190μm, about 200 μm, about 210 μm, about 220 μm, about 230 μm, about 240μm, about 250 μm, about 260 μm, about 270 μm, about 280 μm, about 290μm, about 300 μm, about 310 μm, about 320 μm, about 330 μm, about 340μm, about 350 μm, about 360 μm, about 370 μm, about 380 μm, about 390μm, about 400 μm, about 410 μm, about 420 μm, about 430 μm, about 440μm, about 450 μm, about 460 μm, about 470 μm, about 480 μm, about 490μm, or about 500 μm, or more.

Polymeric micelles and polymeromes are polymeric delivery vehicles withsimilar characteristics to microspheres and can also facilitateencapsulation and matrix integration of the compounds described herein.Fabrication, encapsulation, and stabilization of microspheres for avariety of payloads are within the skill of the art (see, e.g., Vardeand Pack, 2004, supra). The release rate of the microspheres can betailored by type of polymer, polymer molecular weight, copolymercomposition, excipients added to the microsphere formulation, andmicrosphere size. Polymer materials useful for forming microspheresinclude PLA, PLGA, PLGA coated with DPPC, DPPC, DSPC, EVAc, gelatin,albumin, chitosan, dextran, DL-PLG, SDLMs, PEG (e.g., ProMaxx), sodiumhyaluronate, diketopiperazine derivatives (e.g., Technosphere), calciumphosphate-PEG particles, and/or oligosaccharide derivative DPPG (e.g.,Solidose). Encapsulation can be accomplished, for example, using awater/oil single emulsion method, a water-oil-water double emulsionmethod, or lyophilization. Several commercial encapsulation technologiesare available (e.g., ProLease®, Alkerme).

Formulation

The agents and compositions described herein can be formulated by anyconventional manner using one or more pharmaceutically acceptablecarriers or excipients as described in, for example, Remington'sPharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN:0781746736 (2005), incorporated herein by reference in its entirety.Such formulations will contain a therapeutically effective amount of abiologically active agent described herein, which can be in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the subject.

The term “formulation” refers to preparing a drug in a form suitable foradministration to a subject, such as an animal or a human. Thus, a“formulation” can include pharmaceutically acceptable excipients,including diluents or carriers.

The term “pharmaceutically acceptable” as used herein can describesubstances or components that do not cause unacceptable losses ofpharmacological activity or unacceptable adverse side effects. Examplesof pharmaceutically acceptable ingredients can be those havingmonographs in United States Pharmacopeia (USP 29) and National Formulary(NF 24), United States Pharmacopeial Convention, Inc., Rockville, Md.,2005 (“USP/NF”), or a more recent edition, and the components listed inthe continuously updated Inactive Ingredient Search online database ofthe FDA. Other useful components that are not described in the USP/NF,etc., may also be used.

The term “pharmaceutically acceptable excipient,” as used herein, caninclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic, or absorption delaying agents. The useof such media and agents for pharmaceutical active substances is wellknown in the art (see generally Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofaras any conventional media or agent is incompatible with an activeingredient, its use in the therapeutic compositions is contemplated.Supplementary active ingredients can also be incorporated into thecompositions.

A “stable” formulation or composition can refer to a composition havingsufficient stability to allow storage at a convenient temperature, suchas between about 0° C. and about 60° C., for a commercially reasonableperiod of time, such as at least about one day, at least about one week,at least about one month, at least about three months, at least aboutsix months, at least about one year, or at least about two years.

The formulation should suit the mode of administration. The agents ofuse with the current disclosure can be formulated by known methods foradministration to a subject using several routes which include, but arenot limited to, parenteral, pulmonary, oral, topical, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, ophthalmic, buccal, and rectal. The individual agents may alsobe administered in combination with one or more additional agents ortogether with other biologically active or biologically inert agents.Such biologically active or inert agents may be in fluid or mechanicalcommunication with the agent(s) or attached to the agent(s) by ionic,covalent, Van der Waals, hydrophobic, hydrophilic or other physicalforces.

Controlled-release (or sustained-release) preparations may be formulatedto extend the activity of the agent(s) and reduce dosage frequency.Controlled-release preparations can also be used to effect the time ofonset of action or other characteristics, such as blood levels of theagent, and consequently affect the occurrence of side effects.Controlled-release preparations may be designed to initially release anamount of an agent(s) that produces the desired therapeutic effect, andgradually and continually release other amounts of the agent to maintainthe level of therapeutic effect over an extended period of time. Inorder to maintain a near-constant level of an agent in the body, theagent can be released from the dosage form at a rate that will replacethe amount of agent being metabolized or excreted from the body. Thecontrolled-release of an agent may be stimulated by various inducers,e.g., change in pH, change in temperature, enzymes, water, or otherphysiological conditions or molecules.

Agents or compositions described herein can also be used in combinationwith other therapeutic modalities, as described further below. Thus, inaddition to the therapies described herein, one may also provide to thesubject other therapies known to be efficacious for treatment of thedisease, disorder, or condition.

Therapeutic Methods

Also provided is a process of treating connective tissue defect (e.g.,tendon injury or ligament injury) in a subject in need thereof byadministration of a therapeutically effective amount of a compositionincluding Oxo-M and 4-PPBP, and optionally kartogenin or icariin, orboth so as to heal, repair, or regenerate the tendon or ligament.Another process of treating connective tissue involves administering atherapeutically effective amount of Oxo-M, 4PPBP, kartogenin or icariin,or a combination thereof, in a matrix material or scaffold.

Methods described herein are generally performed on a subject in needthereof. A subject in need of the therapeutic methods described hereincan be a subject having, diagnosed with, suspected of having, or at riskfor developing tendon injury or ligament injury. A determination of theneed for treatment will typically be assessed by a history and physicalexam consistent with the disease or condition at issue. Diagnosis of thevarious conditions treatable by the methods described herein is withinthe skill of the art. The subject can be an animal subject, including amammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats,monkeys, hamsters, guinea pigs, and chickens, and humans. For example,the subject can be a human subject.

Generally, a safe and effective amount of a composition including Oxo-Mand 4-PPBP, and optionally kartogenin or icariin, or both, is, forexample, that amount that would cause the desired therapeutic effect ina subject while minimizing undesired side effects. In variousembodiments, an effective amount of a composition including Oxo-M and4-PPBP described herein can recruit PTSCs; induce tenogenicdifferentiation of tendon stem cells, increase (e.g., synergisticallyincrease) tenogenic differentiation and increases expression oftenogenic markers collagen I and II, vimentin, tenomodulin, andscleraxis in PTSCs (e.g., to comparable or higher levels than CTGFstimulation); or heal, repair, or regenerate tendon or ligament defects.

According to the methods described herein, administration can beparenteral, pulmonary, oral, topical, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural,ophthalmic, buccal, or rectal administration.

When used in the treatments described herein, a therapeuticallyeffective amount of a composition including Oxo-M and 4-PPBP andoptionally kartogenin or icariin, or both, can be employed in pure formor, where such forms exist, in pharmaceutically acceptable salt form andwith or without a pharmaceutically acceptable excipient. For example,the compounds of the present disclosure can be administered, at areasonable benefit/risk ratio applicable to any medical treatment, in asufficient amount to recruit PTSCs; induce tenogenic differentiation oftendon stem cells, increase (e.g., synergistically increase) tenogenicdifferentiation and increases expression of tenogenic markers collagen Iand II, vimentin, tenomodulin, and scleraxis in PTSCs (e.g., tocomparable or higher levels than CTGF stimulation); or heal, repair, orregenerate tendon or ligament defects.

The amount of a composition described herein that can be combined with apharmaceutically acceptable carrier to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. It will be appreciated by those skilled in the art thatthe unit content of agent contained in an individual dose of each dosageform need not in itself constitute a therapeutically effective amount,as the necessary therapeutically effective amount could be reached byadministration of a number of individual doses.

Toxicity and therapeutic efficacy of compositions described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals for determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀, (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index that can be expressed as the ratio LD₅₀/ED₅₀,where larger therapeutic indices are generally understood in the art tobe optimal.

The specific therapeutically effective dose level for any particularsubject will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the subject; the time ofadministration; the route of administration; the rate of excretion ofthe composition employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts (see e.g., Koda-Kimble etal. (2004) Applied Therapeutics: The Clinical Use of Drugs, LippincottWilliams & Wilkins, ISBN 0781748453; Winter (2003) Basic ClinicalPharmacokinetics, 4^(th) ed., Lippincott Williams & Wilkins, ISBN0781741475; Shawl (2004) Applied Biopharmaceutics & Pharmacokinetics,McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is wellwithin the skill of the art to start doses of the composition at levelslower than those required to achieve the desired therapeutic effect andto gradually increase the dosage until the desired effect is achieved.If desired, the effective daily dose may be divided into multiple dosesfor purposes of administration. Consequently, single dose compositionsmay contain such amounts or submultiples thereof to make up the dailydose. It will be understood, however, that the total daily usage of thecompounds and compositions of the present disclosure will be decided byan attending physician within the scope of sound medical judgment.

Again, each of the states, diseases, disorders, and conditions,described herein, as well as others, can benefit from compositions andmethods described herein. Generally, treating a state, disease,disorder, or condition includes preventing or delaying the appearance ofclinical symptoms in a mammal that may be afflicted with or predisposedto the state, disease, disorder, or condition but does not yetexperience or display clinical or subclinical symptoms thereof. Treatingcan also include inhibiting the state, disease, disorder, or condition,e.g., arresting or reducing the development of the disease or at leastone clinical or subclinical symptom thereof. Furthermore, treating caninclude relieving the disease, e.g., causing regression of the state,disease, disorder, or condition or at least one of its clinical orsubclinical symptoms. A benefit to a subject to be treated can be eitherstatistically significant or at least perceptible to the subject or to aphysician.

Treatment in accord with the methods described herein can be performedprior to, concurrent with, or after conventional treatment modalitiesfor connective tissue defects, such as tendon or ligament injuries.

A composition including Oxo-M and 4-PPBP can be administeredsimultaneously or sequentially with another agent, such as anantibiotic, an anti-inflammatory, or another agent, or in certainembodiments a cytokine, a growth factor, or even a stem cell. Forexample, a composition including Oxo-M and 4-PPBP can be administeredsimultaneously with another agent, such as an antibiotic or ananti-inflammatory. Simultaneous administration can occur throughadministration of separate compositions, each containing one or more ofa Oxo-M and 4-PPBP, an antibiotic, an anti-inflammatory, or anotheragent. Simultaneous administration can occur through administration ofone composition containing two or more of Oxo-M and 4-PPBP, anantibiotic, an anti-inflammatory, or another agent. A compositionincluding Oxo-M and 4-PPBP can be administered sequentially with anantibiotic, an anti-inflammatory, or another agent. For example, acomposition including Oxo-M and 4-PPBP can be administered before orafter administration of an antibiotic, an anti-inflammatory, or anotheragent.

Administration

Agents and compositions described herein can be administered accordingto methods described herein in a variety of means known to the art. Theagents and composition can be used therapeutically either as exogenousmaterials or as endogenous materials. Exogenous agents are thoseproduced or manufactured outside of the body and administered to thebody. Endogenous agents are those produced or manufactured inside thebody by some type of device (biologic or other) for delivery within orto other organs in the body.

Administration can be parenteral, pulmonary, oral, topical, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, ophthalmic, buccal, or rectal administration.

Agents and compositions described herein can be administered in avariety of methods well known in the arts. Administration can include,for example, methods involving oral ingestion, direct injection (e.g.,systemic or stereotactic), implantation of cells engineered to secretethe factor of interest, drug-releasing biomaterials, polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, implantable matrix devices, mini-osmotic pumps,implantable pumps, injectable gels and hydrogels, liposomes, micelles(e.g., up to 30 μm), nanospheres (e.g., less than 1 μm), microspheres(e.g., 1-100 μm), reservoir devices, a combination of any of the above,or other suitable delivery vehicles to provide the desired releaseprofile in varying proportions. Other methods of controlled-releasedelivery of agents or compositions will be known to the skilled artisanand are within the scope of the present disclosure.

In some embodiments, Oxo-M, 4PPBP, kartogenin or icariin, or combinationthereof, can be mixed with a matrix material (e.g., a surgicaladhesive), and optionally include a visualizing agent as describedbelow. For example, Oxo-M or 4PPBP can be mixed with a fibrin glue, aniCMBA, BioGlue® (e.g., a mix of purified bovine serum albumin (BSA) andglutaraldehyde), or a malonic acid derivative.

A composition described herein can be administered via a scaffoldcomprising a matrix material. In some embodiments, the scaffold caninclude an endogenous or exogenous cell introduced to the scaffold exvivo or in vivo. In some embodiments, the scaffold includes anendogenous progenitor cell. In some embodiments, the scaffold does notinclude an exogenous progenitor cell. In some embodiments, the scaffoldincludes progenitor cell prior to scaffold delivery to the tissue defectsite. In some embodiments, the scaffold does not include a progenitorcell until after scaffold delivery to the tissue defect site. In someembodiments, the scaffold includes an endogenous progenitor cellintroduced to the scaffold in vivo or ex vivo. In some embodiments, thescaffold includes an exogenous progenitor cell introduced to thescaffold in vivo or ex vivo. Features related to cells in the scaffoldcan be combined with other features discussed herein.

In some embodiments, the tissue defect site is at least partiallylocated in an inner or avascular region of a cartilaginous tissue. Insome embodiments, the tissue defect includes a tear, injury,osteoarthritis, or degeneration. In some embodiments, the tissue defectincludes a longitudinal or vertical tear, a radial tear, a horizontaltear, a bucket handle tear, a parrot beak tear, or a flap tear. In someembodiments, the tissue (in which the defect is present) can becartilaginous tissue, cartilage, a meniscus, a knee meniscus, aligament, a ligament enthesis, a tendon, a tendon enthesis, anintervertebral disc, a temporomandibular joint (TMJ), a TMJ ligament, ora triangular fibrocartilage. Features related to tissue and tissuedefect can be combined with other features discussed above and below.

Delivery systems may include, for example, an infusion pump which may beused to administer the agent or composition in a manner similar to thatused for delivering insulin or chemotherapy to specific organs ortumors. Typically, using such a system, an agent or composition can beadministered in combination with a biodegradable, biocompatiblepolymeric implant that releases the agent over a controlled period oftime at a selected site. Examples of polymeric materials includepolyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid,polyethylene vinyl acetate, and copolymers and combinations thereof. Inaddition, a controlled release system can be placed in proximity of atherapeutic target, thus requiring only a fraction of a systemic dosage.

Agents can be encapsulated and administered in a variety of carrierdelivery systems. Examples of carrier delivery systems includemicrospheres, hydrogels, polymeric implants, smart polymeric carriers,and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006)Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-basedsystems for molecular or biomolecular agent delivery can: provide forintracellular delivery; tailor biomolecule/agent release rates; increasethe proportion of biomolecule that reaches its site of action; improvethe transport of the drug to its site of action; allow co-localizeddeposition with other agents or excipients; improve the stability of theagent in vivo; prolong the residence time of the agent at its site ofaction by reducing clearance; decrease the nonspecific delivery of theagent to non-target tissues; decrease irritation caused by the agent;decrease toxicity due to high initial doses of the agent; alter theimmunogenicity of the agent; decrease dosage frequency, improve taste ofthe product; or improve shelf life of the product.

Scaffold Fabrication and Related Embodiments

According to certain embodiments, provided are scaffolds and methods ofmaking scaffolds that incorporate encapsulated Oxo-M, 4-PPBP, kartogeninor Icariin, or any combination thereof. One example is theimplementation of microspheres encapsulating one or more of theforegoing agents and combining them with a matrix material that issuitable for forming a scaffold via 3D printing. A scaffold can then beengineered using this combination of materials. US. Patent Pub.US2018/0085493 provides supporting disclosure for matrix materials,scaffolds produced via 3D printing, polymeric fibers, and methods offabrication, among other information. PCT Pub WO/2016/149153 providessupporting disclosure on methods of treating tissue defects that involvescaffolds that comprise releasable factors that promote migration andproliferation of cells. Such publications are incorporated herein byreference for support of 3D printing techniques and scaffold formation.

A 3D printing device can include one or more cartridges for containing amatrix material, microencapsulated active agent, or various combinationsthereof. A 3D printing device can include one or more cartridges activeconcurrently or sequentially. A 3D printing device can include aplurality of cartridges active concurrently or sequentially. A 3Dprinting device can include a first plurality of cartridges and a secondplurality of cartridges active concurrently or sequentially. Forexample, formation of a 3D printed scaffold described herein can includea plurality of cartridges, each containing independently selected matrixmaterial, microencapsulated active agent, or various combinationsthereof. For example, formation of a 3D printed scaffold describedherein can include 1, 2, 3, 4, 5 6, 7, 8, 9, 10, or more cartridges,each containing independently selected matrix material,microencapsulated active agent, or various combinations thereof.Multiple cartridges can share or have independent elements such as aheating element or printing needle. For example, multiple cartridges caneach contain an independent heating element (e.g., having same ordifferent temperature or melting profiles) or printing needle (e.g.,having same or different inner diameter). A 3D printing device canswitch cartridges before, during or after polymeric microfiber formationor scaffold formation. For example, printing cartridges containing thesame or different matrix materials or microencapsulated active agents orprinting needle size can be switched during fabrication of a polymericmicrofibril or during fabrication of a layer, region, or other structureof the scaffold so as to provide, e.g., differing spatial compositionsof matrix and active agent. As another example, printing cartridgescontaining the same or different matrix materials or microencapsulatedactive agents or printing needle size can be switched to controltemperature of the microencapsulated active agents, e.g., to maintainthe microencapsulated active agents below a threshold temperature (asfurther described herein).

According to one embodiment, provided is a method of forming abiocompatible scaffold. The method involves one or more steps of (i)encapsulating at least one agent in a plurality of microspheres; (ii)combining the plurality of microspheres and a matrix material, thematrix material being suitable for forming a scaffold via 3D printing;(iii) introducing the combination of microspheres and matrix materialinto a first cartridge of a 3D printing device; (iv) heating thecombination of microspheres and matrix material in the first cartridgesufficiently to allow dispensing of the combination while preventingsubstantial degradation of the microsphere or the at least one agentencapsulated in the microsphere; (v) dispensing the heated combinationof microspheres and matrix material from the first cartridge through aprinting needle to form a polymeric microfiber, wherein the microspheresare distributed through the polymeric microfiber; and (vi) forming ascaffold comprising a plurality of the polymeric microfibers, whereinthe microspheres are distributed through the scaffold by way of thepolymeric microfibers.

Another embodiment pertains to a method of forming a polymeric fiberhaving a microencapsulated agent distributed in the polymeric fiber.This method embodiment involves one or more steps of: (i) encapsulatingat least one agent in a plurality of microspheres; (ii) combining theplurality of microspheres and a matrix material, the matrix materialbeing suitable for forming a scaffold via 3D printing; (iii) introducingthe combination of microspheres and matrix material into a firstcartridge of a 3D printing device; (iv) heating the combination ofmicrospheres and matrix material in the first cartridge sufficiently toallow dispensing of the combination of microspheres and matrix materialwhile preventing substantial degradation of the microsphere or the agentencapsulated in the microsphere; and (v) dispensing the heatedcombination of microspheres and matrix material from the cartridgethrough a printing needle to form a polymeric microfiber, wherein themicrospheres are distributed through the polymeric microfiber.

The methods described above may further comprise introducing thecombination of microspheres and matrix material into a second cartridgeof a 3D printing device; heating the combination of microspheres andmatrix material in the second cartridge sufficiently to allow dispensingof the combination of microspheres and matrix material while preventingsubstantial degradation of the microsphere or the agent encapsulated inthe microsphere; and interchanging the first cartridge and the secondcartridge during a printing process.

The at least one agent that is encapsulated in the microspheres includesOxo-M, 4-PPBP, kartogenin or Icariin, or any combination thereof.

Embodiments disclosed herein include microfibers produced by the methodsabove as well as scaffolds made of a plurality of microfibers.

Embodiments of the present invention involve implanting scaffolds astaught herein. Typically, the scaffolds implanted to treat a tissuedefect in a subject in need. Tissue defects treated may be associatedwith a multi-tissue interface. Examples of multi-tissue interfacesinclude, but are not limited to, musculoskeletal system; craniofacialsystem; periodontium; cementum (CM)-periodontal ligament (PDL)-alveolarbone (AB) complex; ligament-to-bone insertion; tendon-to-bone insertion;rotator cuff; supraspinatus tendon-to-bone interface; interface betweentendon, fibrocartilage, or bone; supraspinatustendon-fibrocartilage-bone interface; articular cartilage-to-bonejunction; anterior cruciate ligament (ACL)-to-bone complex; anteriorcruciate ligament-fibrocartilage-bone interface; intervertebral disc;nucleus pulposus-annulus fibrosus-endplates; cementum-periodontalligament-alveolar bone; muscle-to-tendon; inhomogeneous or anisotropictissues; knee meniscus; temporomandibular joint disc; periodontium;root-periodontium complex; synovial joints; or fibrocartilaginoustissues.

In another embodiment, the material provided in any of the cartridges ofthe 3D printing device includes a visualizing agent. Examples ofvisualizing agents include fluorescent molecular probes (e.g.ProSense750, Perkin Elmer; IRDye® 800CW 2-DG, LiCor Biosciences),quantum dots and near-infrared molecular probes, and the like. This willenable the monitoring in vivo degradation of scaffolds made upon theirimplantation into a subject. For example, the visualization agentincludes quantum dots that become embedded into the scaffold. Uponimplantation into the subject the degradation of the scaffold can bemonitored by imaging techniques such as near-infrared imaging, see FIGS.15 and 16. Other examples would include fluorescent molecular probesthat can be detected by fluorescence reflectance imaging.

Screening

Also provided are methods for screening.

The subject methods find use in the screening of a variety of differentcandidate molecules (e.g., potentially therapeutic candidate molecules).Candidate substances for screening according to the methods describedherein include, but are not limited to, fractions of tissues or cells,nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers,ribozymes, triple helix compounds, antibodies, and small (e.g., lessthan about 2000 molecular weight (MW), or less than about 1000 MW, orless than about 800 MW) organic molecules or inorganic molecules,including, but not limited to, salts or metals.

Candidate molecules encompass numerous chemical classes, for example,organic molecules, such as small organic compounds having a molecularweight of more than 50 and less than about 2,500 Daltons. Candidatemolecules can comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group, andusually at least two of the functional chemical groups. The candidatemolecules can comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups.

Candidate molecules can be derivatives of Oxo-M. Candidate molecules canbe derivatives of 4-PPBP.

A candidate molecule can be a compound in a library database ofcompounds. One of skill in the art will be generally familiar with, forexample, numerous databases for commercially available compounds forscreening (see, e.g., ZINC database, UCSF, with 2.7 million compoundsover 12 distinct subsets of molecules; Irwin and Shoichet, J. Chem. Inf.Model 45:177-182, 2005). One of skill in the art will also be familiarwith a variety of search engines to identify commercial sources ordesirable compounds and classes of compounds for further testing (see,e.g., ZINC database; eMolecules.com; and electronic libraries ofcommercial compounds provided by vendors, for example: ChemBridge,Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicalsetc.).

Candidate molecules for screening according to the methods describedherein include both lead-like compounds and drug-like compounds. Alead-like compound is generally understood to have a relatively smallerscaffold-like structure (e.g., molecular weight of about 150 to about350 kD) with relatively fewer features (e.g., less than about 3 hydrogendonors and/or less than about 6 hydrogen acceptors; hydrophobicitycharacter x log P of about −2 to about 4). In contrast, a drug-likecompound is generally understood to have a relatively larger scaffold(e.g., molecular weight of about 150 to about 500 kD) with relativelymore numerous features (e.g., less than about 10 hydrogen acceptorsand/or less than about 8 rotatable bonds; hydrophobicity character x logP of less than about 5) (see, e.g., Lipinski, J. Pharmacol. Toxicol.Methods 44:235-249, 2000). Initial screening can be performed withlead-like compounds.

When designing a lead from spatial orientation data, it can be useful tounderstand that certain molecular structures are characterized as being“drug-like.” Such characterization can be based on a set of empiricallyrecognized qualities derived by comparing similarities across thebreadth of known drugs within the pharmacopoeia. While it is notrequired for drugs to meet all, or even any, of these characterizations,it is far more likely for a drug candidate to meet with clinicalsuccessful if it is drug-like.

Several of these “drug-like” characteristics have been summarized intothe four rules of Lipinski (generally known as the “rules of fives”because of the prevalence of the number 5 among them). While these rulesgenerally relate to oral absorption and are used to predictbioavailability of compound during lead optimization, they can serve aseffective guidelines for constructing a lead molecule during rationaldrug design efforts such as may be accomplished by using the methods ofthe present disclosure.

The four “rules of five” state that a candidate drug-like compoundshould have at least three of the following characteristics: (i) aweight less than 500 Daltons; (ii) a log of P less than 5; (iii) no morethan 5 hydrogen bond donors (expressed as the sum of OH and NH groups);and (iv) no more than 10 hydrogen bond acceptors (the sum of N and Oatoms). Also, drug-like molecules typically have a span (breadth) ofbetween about 8 Å to about 15 Å.

Kits

Also provided are kits. Such kits can include an agent or compositiondescribed herein and, in certain embodiments, instructions foradministration. Such kits can facilitate performance of the methodsdescribed herein. When supplied as a kit, the different components ofthe composition can be packaged in separate containers and admixedimmediately before use. Components include, but are not limited to Oxo-Mand 4-PPBP, and optionally kartogenin and icariin. Such packaging of thecomponents separately can, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the composition. The pack may, for example, comprise metal orplastic foil such as a blister pack. Such packaging of the componentsseparately can also, in certain instances, permit long-term storagewithout losing activity of the components.

Kits may also include reagents in separate containers such as, forexample, sterile water or saline to be added to a lyophilized activecomponent packaged separately. For example, sealed glass ampules maycontain a lyophilized component and in a separate ampule, sterile water,sterile saline or sterile each of which has been packaged under aneutral non-reacting gas, such as nitrogen. Ampules may consist of anysuitable material, such as glass, organic polymers, such aspolycarbonate, polystyrene, ceramic, metal or any other materialtypically employed to hold reagents. Other examples of suitablecontainers include bottles that may be fabricated from similarsubstances as ampules, and envelopes that may consist of foil-linedinteriors, such as aluminum or an alloy. Other containers include testtubes, vials, flasks, bottles, syringes, and the like. Containers mayhave a sterile access port, such as a bottle having a stopper that canbe pierced by a hypodermic injection needle. Other containers may havetwo compartments that are separated by a readily removable membrane thatupon removal permits the components to mix. Removable membranes may beglass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructionalmaterials. Instructions may be printed on paper or other substrate,and/or may be supplied as an electronic-readable medium, such as afloppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, and the like. Detailed instructions may not be physicallyassociated with the kit; instead, a user may be directed to an Internetweb site specified by the manufacturer or distributor of the kit.

Definitions and methods described herein are provided to better definethe present disclosure and to guide those of ordinary skill in the artin the practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

Compositions and methods described herein utilizing molecular biologyprotocols can be according to a variety of standard techniques known tothe art (see, e.g., Sambrook and Russel (2006) Condensed Protocols fromMolecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, ISBN-10: 0879697717; Ausubel, et al. (2002) Short Protocols inMolecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929;Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3ded., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Green andSambrook 2012 Molecular Cloning: A Laboratory Manual, 4th ed., ColdSpring Harbor Laboratory Press, ISBN-10: 1605500569; Elhai and Wolk,Methods Enzymol. 167:747-754, 1988; Studier, Protein Expr. Purif.41:207-234, 2005; Gellissen, ed. (2005) Production of RecombinantProteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH,ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies,Taylor & Francis, ISBN-10: 0954523253).

In some embodiments, numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the present disclosureare to be understood as being modified in some instances by the term“about.” In some embodiments, the term “about” is used to indicate thata value includes the standard deviation of the mean for the device ormethod being employed to determine the value. In some embodiments, thenumerical parameters set forth in the written description and attachedclaims are approximations that can vary depending upon the desiredproperties sought to be obtained by a particular embodiment. In someembodiments, the numerical parameters should be construed in light ofthe number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of some embodiments of thepresent disclosure are approximations, the numerical values set forth inthe specific examples are reported as precisely as practicable. Thenumerical values presented in some embodiments of the present disclosuremay contain certain errors necessarily resulting from the standarddeviation found in their respective testing measurements. The recitationof ranges of values herein is merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range. Unless otherwise indicated herein, each individual value isincorporated into the specification as if it were individually recitedherein.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment(especially in the context of certain of the following claims) can beconstrued to cover both the singular and the plural, unless specificallynoted otherwise. In some embodiments, the term “or” as used herein,including the claims, is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and can also cover other unlisted steps. Similarly, anycomposition or device that “comprises,” “has” or “includes” one or morefeatures is not limited to possessing only those one or more featuresand can cover other unlisted features.

As used herein, substantially, when used as a modifier of a term, refersto a state in which the functional properties of the term are notinfluenced beyond a normal tolerance permitted by one skilled in theart. Thus, a first compound or composition that is substantially free ofa second compound or composition refers to a first compound orcomposition whose functional properties are not influenced by the secondcompound or composition beyond normal tolerance permitted by one skilledin the art.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the present disclosure and does notpose a limitation on the scope of the present disclosure otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of thepresent disclosure.

Groupings of alternative elements or embodiments of the presentdisclosure disclosed herein are not to be construed as limitations. Eachgroup member can be referred to and claimed individually or in anycombination with other members of the group or other elements foundherein. One or more members of a group can be included in, or deletedfrom, a group for reasons of convenience or patentability. When any suchinclusion or deletion occurs, the specification is herein deemed tocontain the group as modified thus fulfilling the written description ofall Markush groups used in the appended claims.

All references cited herein are incorporated by reference in theirentireties for all purposes. Citation of a reference herein shall not beconstrued as an admission that such is prior art to the presentdisclosure.

Having described the present disclosure in detail, it will be apparentthat modifications, variations, and equivalent embodiments are possiblewithout departing the scope of the present disclosure defined in theappended claims. Furthermore, it should be appreciated that all examplesin the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent approaches the inventors have found function well in thepractice of the present disclosure, and thus can be considered toconstitute examples of modes for its practice. However, those of skillin the art should, in light of the present disclosure, appreciate thatmany changes can be made in the specific embodiments that are disclosedand still obtain a like or similar result without departing from thespirit and scope of the present disclosure.

Example 1

The following example shows a combination of Oxo-M and 4-PPBP inducestenogenic differentiation of perivascular tendon stem cells.

Perivascular tendon stem cells (PTSCs) were isolated from patellartendons of 12 week-old Sprague-Dawley rats by sorting cells with strongsurface expression of CD146, following established protocol (Lee, etal., 2015, supra). Culture-expanded PTSC (P2-3) were then treated withselected FAK and ERK1/2 agonists, including Oxotremorine M (Oxo-M) (1mM), PPBP maleate (4-PPBP) (10 μM), Phenylarsine oxide (PAO) (2 μM), and[Tyr4]-Bombesin (BOM) (10 nM), and SKF-83959 (20 μM). A singleconcentration was tested for each molecule as a first screening study.After 1 week, tendon-related mRNA expressions, including collagen I andIII, tenascin-C (Tn-C), vimentin (VIM), tenomodulin (Tnmd), andscleraxis (Scx), were measured.

Results showed that Oxo-M provided significant increases in Tn-C, VIM,and Scx, while 4-PPBP elevated expressions of COL-I and COL-III (FIG.1A-F). When a combination of Oxo-M and 4-PPBP were applied together, thetendon-related gene expressions were dramatically increased up to ˜6-40fold at a similar level achieved by CTGF (FIG. 2A-F).

Other tested FAK and ERK1/2 agonists either did not affect geneexpression or significantly lowered cell viability. BOM and PAO failedto affect the gene expressions. SKF-83959 significantly lowered cellviability.

These findings show that Oxo-M and 4-PPBP have synergetic effects toinduce tenogenic differentiation of the specific population of tendonstem/progenitor cells. These findings also show that not all FAK andERK1/2 agonists necessarily share these properties.

Activation of FAK and ERK1/2 signaling by Oxo-M and 4-PPBP was furtherconfirmed by western blot and siRNA knockdown (KD). By 12 hourstreatment in PTSCs, 4-PPBP resulted in phosphorylation of FAK, whereasOxo-M resulted in phosphorylation of FAK and ERK1/2 (FIG. 3A). Withcombined Oxo-M and 4-PPBP, both P-FAK and P-ERK1/2 were detected (FIG.3A). In contrast, CD146− tendon cells (excluding PTSCs) showed no P-FAKband when treated by Oxo-M, 4-PPBP, and Oxo-M+4-PPBP (FIG. 3A),suggesting that Oxo-M and 4-PPBP selectively target PTSCs. Consistently,the level of tendon-related gene expressions induced by Oxo-M+4-PPBP inCD146− tendon cells was significantly lower than CD146+ PTSCs. Inaddition, siRNA KD of FAK and ERK1/2 were performed as describedpreviously (Lee, et al., 2015, supra), which is hereby incorporatedherein by reference in its entirety for all purposes. Briefly, FAK andERK1/2 were knockdown using Silencer® siRNA (100 nM) and Neon®transfection system (Life Technologies) with pre-optimizedelectroporation conditions (1,400V; 20 mS; 2 pulses). qRT-PCR showedthat FAK and ERK1/2 KD significantly attenuated the Oxo-M+4-PPBP-inducedScx expression in PTSCs (FIG. 3B).

Example 2

This Example shows that delivery in vivo of a combination of Oxo-M and4-PPBP in a rat tendon defect model showed improved tendon healing. Theanimal model was used to study the in vivo response and development ofthese tissues from endogenous cells stimulated by Oxo-M and 4-PPBPdelivery.

This study was designed to investigate effect of Oxo-M and 4-PPBP ontendon healing. Animals were given Oxo-M alone (1 mM), 4-PPBP alone (10μM), and a combination of Oxo-M (1 mM)+4-PPBP (10 μM) along with fibringel as a vehicle after patellar tendon transection. Fibrin alone servedas a vehicle control. Multiple time points (1 and 2 weeks) were selectedto follow up the process of healing and remodeling of tendon. Tissuehealing, matrix formation and remodeling, and functional restorationwere examined using histology, immunohistochemistry, and mechanicaltesting (tensile) as per prior methods (Lee, et al., J. Clin. Invest.120:3340-3349, 2010; Lee, et al., Lancet 376:440-448, 2010; Lee, et al.,Sci. Transl. Med. 6(266):266ra171, 2014; Lee et al., 2015, supra). Eachof these references is incorporated herein in its entirety for allpurposes.

The study was designed as follows:

1. Patella tendon was transected. A 10-mm longitudinal incision was madejust medial to the knee. Upon exposure of patellar tendon, afull-thickness transverse incision was made on the medial part of tendonfrom the inferior pole of the patella.

2. Small molecules (Oxo-M and 4-PPBP, alone or in combination) weredelivered to the transected tendon via fibrin as a delivery vehicle tosee the effect on promoting healing. Fibrin glue, prepared by mixing 1:1of fibrinogen and thrombin with or without Oxotremorine-M (1 mM), PPBPmaleate (10 μM), and Oxo M+4-PPBP was applied on the transection site.

3. Fibrin gel without Oxo-M and 4-PPBP served as a negative controlgroup.

4. At 1 and 2 weeks post-op, the operated animals were euthanizedfollowed by patella tendon collection post mortem for furtherevaluation.

The data demonstrated a novel function of the combined Oxo-M and 4-PPBPin tendon healing. Oxo-M is a non-selective muscarinic acetylcholinereceptor agonist and 4-PPBP is σ1 receptor ligand of which roles intendon or tendon stem cells have never been reported. Although Oxo-M or4-PPBP alone showed only minimal effect, the combination of Oxo-M and4-PPBP showed a notable synergic effect on induced tenogenicdifferentiation of PTSCs and tendon healing.

FIG. 4A-H shows the healing of the fully transected rat patellar tendonsat 1 week post-op after treatment with no small molecules (control; FIG.4A), Oxo-M alone (FIG. 4B), 4-PPBP alone (FIG. 4C) and the combinationof Oxo-M and 4-PPBP (FIG. 4D) and at 2 weeks post-op after treatmentwith no small molecules (control; FIG. 4E), Oxo-M alone (FIG. 4F),4-PPBP alone (FIG. 4G) and the combination of Oxo-M and 4-PPBP (FIG. 4H)by H&E staining. At 1 week post-op, delivery of Oxo-M (1 mM)+4-PPBP (10μM) resulted in reduced gaps and more aligned collagen structure (FIG.4D), as compared to disorganized scar-like tissue with control (FIG.4A), Oxo-M (FIG. 4B), and 4-PPBP (FIG. 4C) alone. At 2 weeks, control(FIG. 4E), Oxo-M (FIG. 4F), and 4-PPBP (FIG. 4G) groups ended up withscar-like healing, whereas Oxo-M+4-PPBP (FIG. 4H) showed significantlyimproved healing with densely aligned fibers.

FIG. 5A-H shows Masson's Trichrome staining of the healing zone oftendons. Delivery of Oxo-M (1 mM)+4-PPBP (10 μM) showed more and densercollagen in the healing zone by 2 weeks (FIG. 5H) as compared todisrupted collagen structure with control (FIG. 5E), Oxo-M (FIG. 5F),and 4-PPBP (FIG. 5G) delivered groups.

FIG. 6A-H shows polarized images of Picrosirius Red stained tendons.Delivery of Oxo-M (1 mM)+4-PPBP (10 μM) showed re-organized collagenfibers by 1 week (FIG. 6D) and 2 weeks (FIG. 6H) as compared todisrupted collagen structure at 1 week and 2 weeks with control (FIG. 6Aand FIG. 6E, respectively), Oxo-M (FIG. 6B and FIG. 6F, respectively),and 4-PPBP (FIG. 6C and FIG. 6G, respectively) delivered groups.

FIG. 7A-D shows immunofluorescence for CD146 and COL-I in tendon healingby small molecules at 1 week post-op. The delivery of the combination ofOxo-M and 4-PPBP (FIG. 7D) into tendon healing significantly increasedthe number of CD146+ PTSCs underdoing differentiation into COL-I+tenocyte-like cells, as compared to control (FIG. 7A), Oxo-M alone (FIG.7B) or 4-PPBP alone (FIG. 7C).

FIG. 8A-F shows collagen fiber orientation assessed by Picrosirius Redstaining with an automated digital image processing for localdirectionality and angular deviation (AD). Alignment of collagen fibersin the Oxo-M+4-PPBP delivered tendon (FIG. 8D) was similar to that ofthe native tendon (FIG. 8E), in contrast to disoriented fibers inscar-like tendon with control (FIG. 8A), Oxo-M alone (FIG. 8B), and4-PPBP alone (FIG. 8C). Quantitatively, the AD of fibers withOxo-M+4-PPBP was significantly smaller than all the other groups (FIG.8F). n=6 per samples, *: p<0.001 compared to control, Oxo-M, and 4-PPBP,#: p<0.05 compared to Oxo-M+4-PPBP.

Since Oxo-M and 4-PPBP are FAK and ERK1/2 agonists, respectively, andERK1/2 is a downstream of FAK signaling, without being limited to anyone theory of the invention the synergic effect is likely attributed tothe potential cross-talk between intracellular signaling mediators. Ascompared to growth factors, small molecules have a number of distinctadvantages including their convenience to use, no cross-contaminationrisk, no immunorejection, and fine-tunable biological effects anddelivery control. Accordingly, the combination of Oxo-M and 4-PPBP,replacing the CTGF's function in tendon healing via selective signalingpathway, overcomes the limitations related to cell and/or proteindelivery for tendon regeneration. The novel combination of Oxo-M and4-PPBP serves as a focused therapeutics for tendon regeneration incomparison with delivery of stem cells, cytokines, or growth factors.

Example 3

This Example shows that delivery in vivo of a combination of Oxo-M and4-PPBP in a rat supraspinatus tendon injury model showed improved tendonhealing. The animal model was used to study the in vivo response anddevelopment of these tissues from endogenous cells stimulated by Oxo-Mand 4-PPBP delivery.

This study was designed to investigate effect of a combination of Oxo-M(1 mM)+4-PPBP (10 μM) along with fibrin gel as a vehicle aftersupraspinatus tendon transection. Fibrin alone served as a vehiclecontrol. Four weeks post-op was selected to follow up the process ofhealing and remodeling of tendon. Tissue healing, matrix formation andremodeling, and functional restoration were examined using histology asper prior methods (Lee, et al., J. Clin. Invest. 120:3340-3349, 2010;Lee, et al., Lancet 376:440-448, 2010; Lee, et al., Sci. Transl. Med.6(266):266ra171, 2014; Lee et al., 2015, supra).

The study was designed as follows:

1. Rat supraspinatus tendon was transected at the tendon enthesis,followed by bone abrasion and suture repair through bone tunnels.

2. Small molecules (Oxo-M and 4-PPBP in combination) were delivered tothe transected tenson via fibrin as a delivery vehicle to see the effecton promoting healing. Fibrin glue, prepared by mixing 1:1 of fibrinogenand thrombin with or without Oxo M+4-PPBP was applied on the transectionsite.

3. Fibrin gel without Oxo-M and 4-PPBP served as a negative controlgroup.

4. At 4 weeks post-op, the operated animals were euthanized followed bysupraspinatus tendon collection post mortem for further evaluation.

The data demonstrated similar results to that found in the rat patellarstudy detailed above. The combination of Oxo-M and 4-PPBP showed anotable synergic effect on tendon healing.

FIG. 9A-I shows native rat supraspinatus tendons at 4× magnification(FIG. 9A), 10× magnification (FIG. 9D) or 20× magnification (FIG. 9G),and the healing of the transected rat supraspinatus tendons at 4 weekspost-op after treatment with fibrin control at 4× magnification (FIG.9B), 10× magnification (FIG. 9E) or 20× magnification (FIG. 9H) or thecombination of Oxo-M and 4-PPBP at 4× magnification (FIG. 9C), 10×magnification (FIG. 9F) or 20× magnification (FIG. 9I). At 4 weeks,Oxo-M+4-PPBP showed significantly improved healing compared to control.

FIG. 10A-I shows polarized images of Picrosirius Red stained native ratsupraspinatus tendons at 4× magnification (FIG. 10A), 10× magnification(FIG. 10D) or 20× magnification (FIG. 10G), and the healing of thetransected rat supraspinatus tendons at 4 weeks post-op after treatmentwith fibrin control at 4× magnification (FIG. 10B), 10× magnification(FIG. 10E) or 20× magnification (FIG. 10H) or the combination of Oxo-Mand 4-PPBP at 4× magnification (FIG. 10C), 10× magnification (FIG. 10F)or 20× magnification (FIG. 10I). Delivery of Oxo-M (1 mM)+4-PPBP (10 μM)showed re-organized collagen fibers as compared to disrupted collagenstructure with control.

FIG. 11A-I shows Masson's Trichrome staining of the healing zone ofnative rat supraspinatus tendons at 4× magnification (FIG. 11A), 10×magnification (FIG. 11D) or 20× magnification (FIG. 11G), and thehealing of the transected rat supraspinatus tendons at 4 weeks post-opafter treatment with fibrin control at 4× magnification (FIG. 11B), 10×magnification (FIG. 11E) or 20× magnification (FIG. 11H) or thecombination of Oxo-M and 4-PPBP at 4× magnification (FIG. 11C), 10×magnification (FIG. 11F) or 20× magnification (FIG. 11I). Delivery ofOxo-M (1 mM)+4-PPBP (10 μM) showed more and denser collagen in thehealing zone by 4 weeks as compared to disrupted collagen structure withcontrol.

FIG. 12A-I shows Safranin O staining of native rat supraspinatus tendonsat 4× magnification (FIG. 12A), 10× magnification (FIG. 12D) or 20×magnification (FIG. 12G), and the healing of the transected ratsupraspinatus tendons at 4 weeks post-op after treatment with fibrincontrol at 4× magnification (FIG. 12B), 10× magnification (FIG. 12E) or20× magnification (FIG. 12H) or the combination of Oxo-M and 4-PPBP at4× magnification (FIG. 12C), 10× magnification (FIG. 12F) or 20×magnification (FIG. 12I). At 4 weeks, Oxo-M+4-PPBP showed significantlyimproved healing compared to control.

Example 4

This Example concerns controlled delivery of a combination of Oxo-M and4-PPBP via poly(lactic-co-glycolic acids) (PLGA) microspheres (μS).

PLGA is a widely used biodegradable polymer for preparation of μS loadedwith small molecules or growth factors (Lee, et al., 2015, supra). PLGAmicrospheres encapsulate small molecules or growth factors by a processcalled “emulsification,” and the encapsulated molecules or factorsrelease in a sustained manner as PLGA slowly degrades via hydrolysis. Assummarized in FIG. 13, the encapsulation efficiency (EE) and the releaserate (RR) of small molecules in PLGA μS are determined by multiplefactors, including the emulsification technique, the size ofmicrospheres, the composition of PLGA and the associated degradationrate, and the hydrophilicity/hydrophobicity of the molecules. The keymicroencapsulation conditions (Table 1) can be tuned to establish a highEE and a prolonged release of Oxo-M and 4-PPBP from PLGA μS forefficient induction of tendon regeneration.

Based on the inventors protocol (Lee, et al., 2014, supra; Lee, et al.,2015, supra; Lee, et al., Tissue Eng. Part A 20:1342-1351, 2014b), Oxo-Mand 4-PPBP can be encapsulated in PLGA μS with the microencapsulationconditions and parameters listed in Table 1.

TABLE 1 Microencapsulation Conditions and Parameters Conditions andExpected Variables Determining Factors Parameters Outcome Size of μSUltra-sonication Frequency: 20 kHz 50 nm to 200 μm Power: 50, 100, 200,in diameter 400 or 600 W Duration: 15 s, 30 s, 1 min, 10 min, 30 minPLGA/solvent 50 mg/mL, 100 concentration mg/mL, 200 mg/mL Release ratePLGA composition Ratio of lactic to 80% release in 2- glycolic acids:50:50, 8 wks in sustained 75:25, 85:15 manner Encapsulation Size of μS,emulsion Derived from above 40-60% of the Efficiency technique, MW, PLGAparameters total applied composition molecules

Briefly, PLGA can be dissolved into chloroform at the above-listedconcentrations, followed by adding 50 μl diluted Oxo-M (1 mM) or 4-PPBP(10 μM) in distilled water (DW). This solution can then be emulsified(primary emulsion) by ultrasonicating to reduce the size of μS. In orderto apply double emulsion for hydrophilic Oxo-M, the primary emulsion(water/oil) can be added to 10 ml 4% (w/v) PVA (poly vinyl alcohol)solution to form the second emulsion (water/oil/water) byultrasonication, followed by 1 min vortexing. The primary-emulsion(4-PPBP) or double-emulsion solution (Oxo-M) can then be added to 250 mlof 0.3% PVA solution followed by continuous stirring for 2 h toevaporate the solvent. Finally, the μS can be filtered, washed with DIwater for 3 times, resuspended in DI water and then lyophilized. Thesize of PLGA μS can be analyzed using SEM (ZEISS SUPRA 55VP) aspreviously described (Lee, et al., 2014, supra; Lee, et al., 2015,supra; Lee, et al., 2014b, supra). The EE can be measured by dissolving10 mg of PLGA μS in chloroform, followed by measuring concentration ofOxo-M or 4-PPBP by High Performance Liquid Chromatography (HPLC). Foranalysis of the release rate, 10 mg of PLGA μS can be incubated in PBSwith a gentle agitation. At the selected time points, the incubationbuffer can be discarded and the amount of Oxo-M or 4-PPBP remained inthe μS can be measured by HPLC as described above to calculate thereleased amounts.

Example 5

This Example concerns in vivo application of the controlled delivery ofa combination of Oxo-M and 4-PPBP via poly(lactic-co-glycolic acids)(PLGA) microspheres (μS) for tendon regeneration.

Once establishing a set of PLGA microencapsulation conditions andresulted EE and release rates, PLGA μS-loaded with Oxo-M and 4-PPBP withslow, intermediate, and fast release rates can be applied to the in vivotendon healing model detailed above. Various doses of PLGA μS (10, 50,100 mg/mL) with different release rates can be delivered via a fibringel into fully transected rat patella tendon (PT) as detailed above.Briefly, a 10-mm longitudinal incision can be made just medial to theknee. Upon exposure of the patellar tendon, a full-thickness transverseincision can be made using a No. 11 blade scalpel. Fibrin gel, preparedby mixing 1:1 of fibrinogen (50 mg/mL) and thrombin (50 U/mL) with orwithout selected small molecules at the optimized doses can be appliedon the transection site using Fibrijet® dual injector. A 2-0 Ethibondsuture (Ethicon Inc, Somerville, N.J., USA) can then be passed throughthe tibia and quadriceps in a cerclage technique. The surgical site canthen be closed using 4.0 absorbable (continuous stitch) for thesubcutaneous layer and 4.0 PDS and monocryl (interrupted stitches) forthe skin closure. At multiple time points (1-4 wks), animals can beeuthanized and the quality of tendon healing in association withendogenous PTSCs can be analyzed as described in Example 2 above.

Statistical comparisons between the control and the small molecules(±μS) group can be made for all the data available for quantification,including the digitally analyzed collagen orientation under polarizedimages (angular deviation: AD), the ratio and number ofmarkers-expressing PTSCs, and mechanical properties. At least 10biological replicates, based on the Power analysis below, can beincluded for all the statistical analyses of the outcome from randomlyselected tissue sections as well as tissue samples for mechanical tests.CTGF delivery can be used as a positive control for the in vivo studies.

As detailed above, a series of mechanical tests can be performed forfunctional characterization of the regenerated tendons, usingElectroforce® Biodynamic® test system (Bose Corp., Eden Prairie, Minn.)following established protocols for testing tendon's mechanicalproperties as detailed above (Lee, et al., 2015, supra). Themuscle-tendon-bone complexes can be harvested and prepared as described(Lee, et al., 2015, supra), clamped with tensile jigs, andpreconditioned for 10 cycles at 0.1 Hz between 5N and 10N as maintaining100% humidity. For tensile properties, a constant displacement rate at0.25 mm/sec can be applied until failure. Elongation can be measured bythe embedded displacement sensor and a Digital Video Extensometer (DVE),and force curve can be recorded via the embedded load-cell. Fromforce-displacement curve, stiffness, maximum force, failure displacementcan be calculated. For stress-relaxation test, a load of 30N can beapplied and the final displacement can be held constant while thedeclining load is measured at 4 Hz either for 15 minutes or until theload changed less than 0.1 percent over 1 minute, and peak andrelaxation moduli and coefficient of relaxation can be calculated.

Example 5

This Example concerns in vivo application of the controlled delivery ofa combination of Oxo-M and 4-PPBP via poly(lactic-co-glycolic acids)(PLGA) microspheres (μS) in tendinopathy models.

Upon confirmation of the improved healing of transected rat PT,controlled co-delivery of Oxo-M and 4-PPBP can be applied forpathological tendon models that more closely replicate the chroniceffects of tendon injury/disease. Pathological conditions have beencreated in tendons at multiple anatomical locations (e.g., PT, rotatorcuff, and Achilles) by mechanical overuse (Lake, et al., Disabil.Rehabil. 30:1530-1541, 2008; Lui, et al., Scand. J. Med. Sci. Sports21:3-17, 2011) or genetic modifications. The overuse-inducedtendinopathy in rat PT can be adopted first, featured byhypercellularity, disorganized collagen fibers, increased cartilaginousmatrix, decreased mechanical properties, elevated inflammation and MMPactivities, and impaired healing upon acute injury. The tendinopathy canbe induced in 30 week-old SD rats by a repetitive exercise protocol thatconsists of treadmill running at 17 m/min on a 10° decline for 1hour/day, 5 days/week using a rodent treadmill (Exer-3/6 OpenTreadmill). After 4-6 weeks of treadmill running, pathological changesin the PT can be evaluated by histology/immunohistochemistry andbiochemical assays. To determine effect of Oxo-M and 4-PPBP onprevention of tendon pathology, peritendinous injection of Oxo-M+4-PPBPμS can be made into the tendons with a 27 gauge needle and a 100 μLsyringe prior to starting the treadmill exercise. Control animals canreceive saline injections with empty μS in the same volume. Then aseries of measurements can be made to determine effects of theOxo-M+4-PPBP μS injections on the progress of tendinopathy, the numberand bioactivities of PTSCs, the expression patterns of pro- andanti-inflammatory cytokines (IL-1β, IL-6, IL-10), MMPs (-1, -3, -8, -9)and TIMPs (-1, -2, -3, -4), and mechanical properties of tendons asdescribed above. In parallel, Oxo-M+4-PPBP μS can also be applied forhealing of pathological tendons that has further-impaired healingcapacity. Briefly, tendons that undergo mechanical overuse for 6 weekscan be fully transected, followed by repair with cerclage suture. ThenOxo-M+4-PPBP μS can be delivered to the repaired tendons via fibrin gelas described above. By 1-6 weeks post-op, the quality of tendon healingin association with endogenous PTSCs can be analyzed as described inExample 2 above.

All quantitative data in the in vitro and in vivo experiments can besubjected to statistical analysis. For normal data distribution, theAnalysis of Variance (ANOVA) with Bonferroni tests can be used. Forskewed data distribution, nonparametric tests such as Kruskal-Wallisanalysis of variance can be used with α=0.05. Power analysis: Samplenumbers for all the in vitro and in vivo experiments can be determinedby Power analysis with a significance level of 0.05 and effect size of1.50. Estimated outcome for power analysis can be adopted as describedfor soft tissue regeneration (Lee, et al., 2014, supra). Sample numbersof 8 and 10 per group can be estimated from power analysis for in vitroand in vivo experiments, respectively.

1-18. (canceled)
 19. A composition comprising: a polymeric microfiberproduced by 3D printing; and a plurality of microspheres encapsulatingat least one agent; wherein the microspheres are distributed through thepolymeric microfiber and wherein the at least one agent comprises Oxo-M,4-PPBP, kartogenin or Icariin, or any combination thereof.
 20. Thecomposition of claim 19, wherein the microspheres comprise at least afirst group of microspheres and a second group of microspheres; thefirst group of microspheres and the second group of microspherescomprise at least one agent; and the first group of microspheres and thesecond group of microspheres comprise at least one different agent. 21.The composition of claim 19, wherein the polymeric microfiber comprisespolycarprolactone (PCL).
 22. The composition of claim 19, furthercomprising a visualizing agent.
 23. The composition of claim 22, whereinvisualization agent is detectable with near infrared imaging.
 24. Thecomposition of claim of claim 22, wherein the visualizing agent is aquantum dot. 25-35. (canceled)